Project Summary/Abstract Adverse consequences from having a faulty circadian clock include compromised sleep quality, poor performance, and increased risk for accidents in the short-term, and metabolic diseases and cancer in the long-term. However, our understanding of circadian sleep disorders?and thus our ability to develop treatments for them?is limited by the incompleteness of our molecular models and our dearth of animal models. For example, many patients with circadian sleep disorders have wake-sleep cycles that shift daily in an unpredictable manner. Yet there were no animal models that emulated such unstable rhythms until our laboratory developed one in the last two years. There may be other human circadian disorders that go unrecognized or are poorly understood because our limited set of animal models fall far short of matching the diversity of the human gene pool. We propose to study a highly diverse set of mutations and single-nucleotide polymorphisms (SNPs) to elucidate their effect on circadian clock function. We would thus enhance the understanding of diverse chronotypes and sleep disorders in humans and pave the way for developing effective treatments. Specific Aim 1: Identify genetic variations in PER that may be associated with human circadian sleep disorders. Because it would be prohibitively expensive to use animal models to recapitulate all known SNPs and mutations in human clock genes, we will first characterize a large number of important variants in cell culture, using U2OS and MEFs, cell lines widely accepted as models for circadian rhythms. Our focus will be on SNPs or mutations in Period (Per) genes because Per1 and 2 are the most important mammalian genes in determining the clock?s period and phase, and hundreds of variants of each gene are known to exist. We will use the CRISPR/Cas9 technology to reproduce diverse variants in clock cells, and we will evaluate their circadian phenotypes. We have already identified a Per1 deletion mutant revealing a novel motif critical for phosphorylation, leading us to a novel hypothesis for how PER phosphorylation is dynamically regulated. We will test this hypothesis and continue to study other mechanisms of posttranslational regulation critical for the clock. We will also reproduce a select set of variants in mice for in vivo testing. Specific Aim 2: Identify key regulators of robust and timed proteasomal degradation of PER. Our previous and ongoing studies suggest that the circadian clock is dependent on rhythms of the core clock protein PER, and its proteasomal degradation is regulated by both ubiquitination and deubiquitination. We propose to conduct a systematic search for E3 ligases that regulate PER ubiquitination and degradation, and to test their function in cell models, along with the functions of candidate deubiquitnases. In Aim 1, we will identify key regulatory sites within PER, while in Aim 2, we will identify key enzymes involved in that regulation.